EGFR-mutant Lung Cancer Cells Research Articles

Abstract 1192: Increased regulatory T cells induced by glycolytic metabolic change in EGFR mutant NSCLC after EGFR TKI therapy

Abstract Background. Studies on the immune microenvironment of EGFR mutant lung cancer have been limited. We analyzed the effect of immune microenvironments on the development of EGFR-TKI resistance in EGFR-mutated lung cancer. Methods. The EGFR mutant lung cancer cell line was co-cultured with activated PBMC for 72 hours with EGFR-TKI. Changes of cytokines/chemokines in the media, PD-1 expression of CD8+ T cells, regulatory T cells fraction and transcriptome analysis of tumor cells were analyzed. We also performed immune profile analysis of fresh tissues of 21 surgically resected NSCLC (7 EGFR mutant and 14 EGFR wild) by multicolor FACS. Results. The Cytotoxicity of EGFR-TKI (elrotinib) in EGFR mutant HCC827 and H4006 lung cancer cell lines co-cultured with active PBMC tended to decrease. IL-6, IL-8, VEGF, TGF-B1, CXCL1, and CXCL10 were significantly increased after co-culture with activated PBMC but did not decrease after addtional EGFR-TKI treatment. IFN gamma increased after co-culture with activated PBMC but decreased after EGFR-TKI treatment. PD-L1 expression on tumor cells increased after co-culture with activated PBMC (p = 0.08 in HCC827 and p = 0.09 in H4006) but did not decrease after co-culture with activated PBMC and EGFR-TKI treatment (p = 0.36 in HCC827 and p = 0.45 in H4006). PD-L1 expression of A549 (EGFR wild type) did not change with co-culture or EGFR-TKI treatment. PD-1 expression of CD8 T cell co-cultured with HCC827 or H4006 did not change, however proportion of regulatory T cell increased after co-culture with HCC827 or H4006 (p=0.05 and p=0.08, respectively) and did not decrease during co-culture and EGFR-TKI treatment. Proportion of regulatory T cell in co-cultures with A549 or H1975 (erltinib resistant cell line) did not change during co-culture or EGFR-TKI treatment. Transcriptome analysis by RNA sequencing showed 1747 gene sets were differentially expressed in EGFR-TKI treated EGFR mutant cell line co-cultured with activated PBMC compared to EGFR-TKI treatment alone. Interferon gamma response pathway (NES 2.65, FDR q<0.1) and glycolytic pathway (NES 2.15, FDR<0.1) were most significantly changed. Immune profile analysis of human EGFR mutant lung cancer showed that CD4+/CD3+ T cells in EGFR mutant groups was increased compared to EGFR wild group. Proportion of FOXP3+CD25+CD4+ T reg in EGFR mutant group tended to increase compared to EGFR wild group (1.352±0.4 vs 0.74 ± 0.16%, p=0.256). Conclusion. The regulator T cell is considered to be important in EGFR mutant NSCLC in induction of immuno suppressive microenvrionement and EGFR-TKI resistance. Study on glycolytic pathway mediated immuno suppressive microenvionment and EGFR-TKI resistance is currently ongoing Citation Format: Sook-hee Hong, Nahyeon Kang, Seung joon Kim, Okrane Kim, Jin-hyoung Kang, Sook Whan Sung. Increased regulatory T cells induced by glycolytic metabolic change in EGFR mutant NSCLC after EGFR TKI therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1192.

Abstract 4762: Activation of AXL as a preclinical acquired resistance mechanism against osimertinib treatment in EGFR-mutant non-small cell lung cancer cells

Abstract Osimertinib (AZD9291) has an efficacy superior to that of standard EGFR-tyrosine kinase inhibitors for the first-line treatment of EGFR-mutant advanced non-small cell lung cancer (NSCLC) patients. However, patients treated with osimertinib eventually acquire drug resistance, and novel therapeutic strategies to overcome acquired resistance are needed. In clinical or preclinical models, several mechanisms of acquired resistance to osimertinib have been elucidated. However, the acquired resistance mechanisms when osimertinib is initially used for EGFR-mutant NSCLC remain unclear. In this study, we experimentally established acquired osimertinib-resistance cell lines from EGFR-mutant NSCLC cell lines and investigated the molecular profiles of resistant cells to uncover the mechanisms of acquired resistance. Various resistance mechanisms were identified, including the acquisition of MET amplification, EMT induction, and the upregulation of AXL. Using targeted next-generation sequencing with a multi-gene panel, no secondary mutations were detected in our resistant cell lines. Among three MET-amplified cell lines, one cell line was sensitive to a combination of osimertinib and crizotinib. Acquired resistance cell lines derived from H1975 harboring the T790M mutation showed AXL upregulation, and the cell growth of these cell lines was suppressed by a combination of osimertinib and cabozantinib, an inhibitor of multiple tyrosine kinases including AXL, both in vitro and in vivo. Our results suggest that AXL might be a therapeutic target for overcoming acquired resistance to osimertinib. Citation Format: Kei Namba, Kazuhiko Shien, Yuta Takahashi, Hiroki Sato, Takahiro Yoshioka, Ken Suzawa, Hiromasa Yamamoto, Junichi Soh, Shuta Tomida, Shinichi Toyooka. Activation of AXL as a preclinical acquired resistance mechanism against osimertinib treatment in EGFR-mutant non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4762.

Abstract 698: Enhancing apoptosis to improve the therapy of EGFR-mutant lung cancer

Abstract EGFR-TKIs have shown clinical efficacy in EGFR-mutant lung cancer patients, but most of the cases eventually develop resistance to the drugs, including the third-generation EGFR-TKI. Here, we develop a cell death mechanism-based combination strategy to enhance the therapeutic effect of EGFR-TKIs as well as to overcome the acquired resistance to EGFT-TKI in EGFR-mutant lung cancer. Through high-throughput screening of a custom pathway inhibitor library (-1000 drugs), we discovered Aurora kinase inhibitors that synergized with a third-generation EGFR-TKI (AZD9291) to induce apoptosis in EGFR-mutant lung cancer cells. Aurora kinase inhibitors enhanced AZD9291-mediated induction of BIM and PUMA, two important sentinels interconnecting the EGFR signal transduction pathway and the mitochondrial apoptotic machinery BAX and BAK. Combined inhibition of Aurora kinases and EGFR prevented the emergence of EGFR-TKI resistant clones in clonogenic assays. Interestingly, AZD9291 resistant lung cancer cells exhibited greater sensitivity to Aurora kinase inhibitors compared with parental cells. In conclusion, we found that the combination of AZD9291 and Aurora kinase inhibitors synergistically induced BIM and PUMA-mediated apoptosis, which may render a promising strategy to improve the therapy of EGFR-mutant lung cancer. <!–EndFragment–> Citation Format: Kosuke Tanaka, Song Han, Yogesh T. Ganesan, James J. Hsieh, Emily H. Cheng. Enhancing apoptosis to improve the therapy of EGFR-mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 698.

Abstract 1273: Molecular dynamics prediction of the mechanism of acquired resistance to EGFR inhibitors in EGFR-mutant lung cancer

Abstract Background Acquired drug resistance is the most challenging problem in treating nonsmall cell lung cancer (NSCLC) patients with EGFR tyrosine kinase inhibitor (TKI). We evaluated whether resistance cell subclones significantly increased shortly after treatment with EGFR-TKIs could determine the final resistance phenotype for EGFR-TKIs in the tumor with EGFR mutations. Methods We generated 2 EGFR-mutant lung cancer cell lines (PC9GR and HCC827GR) resistant to gefitinib and evaluated the resistance mechanisms in each cell line. In parent cell lines (PC9 and HCC827) the amounts of EGFR T790M mutation and C-MET and VIMENTIN mRNA expression were measured before and after 48 hours treatment with gefitinib or paclitaxel. Using a cell line derived directly from tumors of treatment-naïve NSCLC patient with EGFR mutation, the above tests were repeated. We prospectively collected the bloods from 4 NSCLC patients with EGFR mutations at baseline and eight weeks after EGFR-TKI treatment and measured the level of T790M mutation in circulating tumor DNAs (ctDNA) from their bloods by colourimetric assay. Results EGFR T790M mutation was detected in PC9GR cells whereas C-MET amplification in HCC827GR cells. The proportion of EGFR T790M mutation significantly increased in the PC9 cells treated with gefitinib whereas not in the same cells treated with paclitaxel or not in the HCC827 treated with gefitinib or paclitaxel. The mRNA expression of C-MET was significantly increased in the HCC827 treated with gefitinib. The VIMENTIN mRNA expression was not changed in both cell lines. When we treated the patient-derived cells with gefitnib, the frequency of T790M significantly increased whereas C-MET mRNA expression decreased and VIMENTIN mRNA expression was not changed. In the corresponding resistance tumor tissue, only T790M mutation was detected without C-MET amplification and VIMENTIN overexpression. Two cases whose T790M mutation level in their ctDNAs increased at 8 weeks by more than 50% revealed T790M mutation in their final resistance tumor tissue. But, in other cases without an early surge of T790M mutation in their ctDNAs, T790M mutation was not detected in posttreatment biopsy sample. Conclusions Early-on-treatment quantitative change of resistance-related gene alterations may predict the final mechanism of resistance to EGFR-TKI. Citation Format: Youngjoo Lee, Yu-Ra Choi. Molecular dynamics prediction of the mechanism of acquired resistance to EGFR inhibitors in EGFR-mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1273.

Abstract 313: AXL confers intrinsic resistance to osimertinib and the emergence of tolerant cells

Abstract A novel EGFR-tyrosine kinase inhibitor (TKI), osimertinib, has marked efficacy in patients with EGFR-mutated lung cancer. However, some patients show intrinsic resistance and an insufficient response to osimertinib. This study showed that osimertinib adversely stimulated AXL by inhibiting a negative feedback loop. Activated AXL was associated with EGFR and HER3 in maintaining cell survival and inducing the emergence of cells tolerant to osimertinib. AXL inhibition reduced the viability of EGFR-mutated lung cancer cells overexpressing AXL that were exposed to osimertinib. The addition of an AXL inhibitor during either the initial or tolerant phases reduced tumor size and delayed tumor re-growth compared to osimertinib alone. AXL was highly expressed in clinical specimens of EGFR-mutated lung cancers and its high expression was associated with a low response rate to EGFR-TKI. These results indicated pivotal roles for AXL and its inhibition in the intrinsic resistance to osimertinib and the emergence of osimertinib-tolerant cells. Citation Format: Hirokazu Taniguchi, Tadaaki Yamada, Rong Wang, Keiko Tanimura, Akihiro Nishiyama, Azusa Tanimoto, Yuta Adachi, Shinji Takeuchi, Hiroyuki Yamaguchi, Minoru Fukuda, Koichi Takayama, Hiroshi Mukae, Seiji Yano. AXL confers intrinsic resistance to osimertinib and the emergence of tolerant cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 313.

Role of FBXW7 in the quiescence of gefitinib-resistant lung cancer stem cells in EGFR-mutant non-small cell lung cancer.

Several recent studies suggest that cancer stem cells (CSCs) are involved in intrinsic resistance to cancer treatment. Maintenance of quiescence is crucial for establishing resistance of CSCs to cancer therapeutics. F-box/WD repeat-containing protein 7 (FBXW7) is a ubiquitin ligase that regulates quiescence by targeting the c-MYC protein for ubiquitination. We previously reported that gefitinib-resistant persisters (GRPs) in EGFR-mutant non-small cell lung cancer (NSCLC) cells highly expressed octamer-binding transcription factor 4 (Oct-4) as well as the lung CSC marker CD133, and they exhibited distinctive features of the CSC phenotype. However, the role of FBXW7 in lung CSCs and their resistance to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors in NSCLC is not fully understood. In this study, we developed GRPs from the two NSCLC cell lines PC9 and HCC827, which express an EGFR exon 19 deletion mutation, by treatment with a high concentration of gefitinib. The GRPs from both PC9 and HCC827 cells expressed high levels of CD133 and FBXW7, but low levels of c-MYC. Cell cycle analysis demonstrated that the majority of GRPs existed in the G0/G1 phase. Knockdown of the FBXW7 gene significantly reduced the cell number of CD133-positive GRPs and reversed the cell population in the G0/G1-phase. We also found that FBXW7 expression in CD133-positive cells was increased and c-MYC expression was decreased in gefitinib-resistant tumors of PC9 cells in mice and in 9 out of 14 tumor specimens from EGFR-mutant NSCLC patients with acquired resistance to gefitinib. These findings suggest that FBXW7 plays a pivotal role in the maintenance of quiescence in gefitinib-resistant lung CSCs in EGFR mutation-positive NSCLC.

Cause-and-Effect relationship between FGFR1 expression and epithelial-mesenchymal transition in EGFR-mutated non-small cell lung cancer cells

ObjectivesIncreased FGFR1 expression is associated with resistance to tyrosine kinase inhibitors (TKIs) in EGFR-mutated NSCLC cells and often concomitant with epithelial to mesenchymal transition (EMT). However, the cause-and-effect relationship between increased FGFR1 expression and EMT in the genetic background of EGFR-mutated non-small cell lung cancer (NSCLC) cells is not clear. Previous studies have specifically addressed the relationship between EMT and increased FGFR1 expression in the context of simultaneous TKI-mediated blocking of EGFR-signaling. Here, in the context of EGFR-mutated NSCLC cells with active EGFR-signaling, we have examined whether increased FGFR1 expression drives EMT or is an EMT passenger event. Materials and methodsFor cause-and-effect analyses between EMT and FGFR1 expression, including expression of alternative spliced FGFR1 isoforms, we used CRISPR-dCAS9-SAM-mediated induction of the endogenous FGFR1 and ZEB1 genes, as well as biochemical EMT-induction, in PC9 and HCC827 NSCLC cell lines harboring activating EGFR-mutations. ResultsWe find that FGFR1 expression correlates with a ZEB1-associated EMT gene expression profile in NSCLC cells. In experiments using NSCLC cell lines harboring activating EGFR-mutations we show that CRISPR-dCAS9-SAM-mediated induction of FGFR1 expression is neither driving an increase in ZEB1 expression nor EMT characteristics. However, CRISPR-dCAS9-SAM-mediated induction of ZEB1 expression drives EMT characteristics and an increase in FGFR1 expression. Biochemical induction of EMT also drives an increase in FGFR1 expression. ConclusionFrom our findings concerning the cause-and-effect relationship in the genetic background of EGFR-mutated NSCLC cells, we conclude that an increase in ZEB1 expression is a driver of EMT resulting in concomitant increased FGFR1 expression, whereas an increase in FGFR1 expression is insufficient to drive concomitant EMT.

MiR-147b-mediated TCA cycle dysfunction and pseudohypoxia initiate drug tolerance to EGFR inhibitors in lung adenocarcinoma.

Drug-tolerance is an acute defense response prior to a fully drug-resistant state and tumor relapse, however there are few therapeutic agents targeting drug-tolerance in the clinic. Here we show that miR-147b initiates a reversible tolerant-state to the EGFR inhibitor osimertinib in non-small cell lung cancer. With miRNA-seq analysis we find that miR-147b is the most upregulated microRNA in osimertinib-tolerant and EGFR mutated lung cancer cells. Whole transcriptome analysis of single-cell derived clones reveals a link between osimertinib-tolerance and pseudohypoxia responses irrespective of oxygen levels. Further metabolomics and genetic studies demonstrate that osimertinib-tolerance is driven by miR-147b repression of VHL and succinate dehydrogenase linked to the tricarboxylic acid cycle and pseudohypoxia pathways. Finally, pretreatment with a miR-147b inhibitor delays osimertinib-associated drug tolerance in patient-derived three-dimensional (3D) structures. This link between miR-147b and tricarboxylic acid cycle may provide promising targets for preventing tumor relapse.

Ganetespib in Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor-resistant Non-small Cell Lung Cancer.

The 90-kDa heat-shock protein (HSP90) is a chaperone protein expressed at high levels in cancer cells and is involved in the folding or stabilization of several client proteins, including epidermal growth factor receptor (EGFR). Ganetespib is a second-generation HSP90 inhibitor with a potent antitumor effect against various cancer types. This study examined the antitumor effect of ganetespib in EGFR-mutant non-small cell lung cancer (NSCLC) cells and experimentally established EGFR-tyrosine kinase inhibitor (TKI)-resistant cells harboring various resistance mechanisms, including EGFR T790M mutation, met proto-oncogene amplification, and epithelial-mesenchymal transition. Ganetespib showed a potent antitumor effect at low concentrations, suppressing EGFR-related downstream pathway molecules and inducing cleavage of poly ADP-ribose polymerase in all examined EGFR-TKI-resistant cell lines in vitro. Ganetespib also inhibited in vivo tumor growth in resistant cells harboring EGFR T790M. Ganetespib might be a promising therapeutic option for the treatment of patients with EGFR-TKI-resistant NSCLC.

Expression of Concern: Autophagosome-Mediated EGFR Down-Regulation Induced by the CK2 Inhibitor Enhances the Efficacy of EGFR-TKI on EGFR-Mutant Lung Cancer Cells with Resistance by T790M.

Expression of Concern: Autophagosome-Mediated EGFR Down-Regulation Induced by the CK2 Inhibitor Enhances the Efficacy of EGFR-TKI on EGFR-Mutant Lung Cancer Cells with Resistance by T790M.

Acquired BRAF Rearrangements Induce Secondary Resistance to EGFR therapy in EGFR-Mutated Lung Cancers

IntroductionMultiple genetic mechanisms have been identified in EGFR-mutant lung cancers as mediators of acquired resistance (AR) to EGFR tyrosine kinase inhibitors (TKIs), but many cases still lack a known mechanism. MethodsTo identify novel mechanisms of AR, we performed targeted large panel sequencing of samples from 374 consecutive patients with metastatic EGFR-mutant lung cancer, including 174 post-TKI samples, of which 38 also had a matched pre-TKI sample. Alterations hypothesized to confer AR were introduced into drug-sensitive EGFR-mutant lung cancer cell lines (H1975, HCC827, and PC9) by using clustered regularly interspaced short palindromic repeats/Cas9 genome editing. MSK-LX138cl, a cell line with EGFR exon 19 deletion (ex19del) and praja ring finger ubiquitin ligase 2 gene (PJA2)/BRAF fusion, was generated from an EGFR TKI–resistant patient sample. ResultsWe identified four patients (2.3%) with a BRAF fusion (three with acylglycerol kinase gene (AGK)/BRAF and one with PJA2/BRAF) in samples obtained at AR to EGFR TKI therapy (two posterlotinib samples and two posterlotinib and postosimertinib samples). Pre-TKI samples were available for two of four patients and both were negative for BRAF fusion. Induction of AGK/BRAF fusion in H1975 (L858R + T790M), PC9 (ex19del) and HCC827 (ex19del) cells increased phosphorylation of BRAF, MEK1/2, ERK1/2, and signal transducer and activator of transcription 3 and conferred resistance to growth inhibition by osimertinib. MEK inhibition with trametinib synergized with osimertinib to block growth. Alternately, a pan-RAF inhibitor as a single agent blocked growth of all cell lines with mutant EGFR and BRAF fusion. ConclusionBRAF fusion is a mechanism of AR to EGFR TKI therapy in approximately 2% of patients. Combined inhibition of EGFR and MEK (with osimertinib and trametinib) or BRAF (with a pan-RAF inhibitor) are potential therapeutic strategies that should be explored.

Activation of AXL as a Preclinical Acquired Resistance Mechanism Against Osimertinib Treatment in EGFR-Mutant Non-Small Cell Lung Cancer Cells.

Osimertinib (AZD9291) has an efficacy superior to that of standard EGFR-tyrosine kinase inhibitors for the first-line treatment of patients with EGFR-mutant advanced non-small cell lung cancer (NSCLC). However, patients treated with osimertinib eventually acquire drug resistance, and novel therapeutic strategies to overcome acquired resistance are needed. In clinical or preclinical models, several mechanisms of acquired resistance to osimertinib have been elucidated. However, the acquired resistance mechanisms when osimertinib is initially used for EGFR-mutant NSCLC remain unclear. In this study, we experimentally established acquired osimertinib-resistant cell lines from EGFR-mutant NSCLC cell lines and investigated the molecular profiles of resistant cells to uncover the mechanisms of acquired resistance. Various resistance mechanisms were identified, including the acquisition of MET amplification, EMT induction, and the upregulation of AXL. Using targeted next-generation sequencing with a multigene panel, no secondary mutations were detected in our resistant cell lines. Among three MET-amplified cell lines, one cell line was sensitive to a combination of osimertinib and crizotinib. Acquired resistance cell lines derived from H1975 harboring the T790M mutation showed AXL upregulation, and the cell growth of these cell lines was suppressed by a combination of osimertinib and cabozantinib, an inhibitor of multiple tyrosine kinases including AXL, both in vitro and in vivo. Our results suggest that AXL might be a therapeutic target for overcoming acquired resistance to osimertinib. IMPLICATIONS: Upregulation of AXL is one of the mechanisms of acquired resistance to osimertinib, and combination of osimertinib and cabozantinib might be a key treatment for overcoming osimertinib resistance.

AXL confers intrinsic resistance to osimertinib and advances the emergence of tolerant cells

A novel EGFR-tyrosine kinase inhibitor (TKI), osimertinib, has marked efficacy in patients with EGFR-mutated lung cancer. However, some patients show intrinsic resistance and an insufficient response to osimertinib. This study showed that osimertinib stimulated AXL by inhibiting a negative feedback loop. Activated AXL was associated with EGFR and HER3 in maintaining cell survival and inducing the emergence of cells tolerant to osimertinib. AXL inhibition reduced the viability of EGFR-mutated lung cancer cells overexpressing AXL that were exposed to osimertinib. The addition of an AXL inhibitor during either the initial or tolerant phases reduced tumor size and delayed tumor re-growth compared to osimertinib alone. AXL was highly expressed in clinical specimens of EGFR-mutated lung cancers and its high expression was associated with a low response rate to EGFR-TKI. These results indicated pivotal roles for AXL and its inhibition in the intrinsic resistance to osimertinib and the emergence of osimertinib-tolerant cells.

The role of histone lysine demethylases in cancer cells' resistance to tyrosine kinase inhibitors.

Current cancer therapies are often associated with treatment failure and reduced patients’ survival due to drug resistance. There are various mechanisms involved in the acquisition of cancer drug resistance, including the selection of advantageous mutations, overexpression of transporter proteins and epigenetic alterations. In this context, epigenetic alterations refer to chromatin-mediated regulation of gene expression that results in heritable changes in the cellular phenotype. There is an ever-growing body of evidence suggesting that epigenetic mechanisms play an important role in bringing about drug resistance in cancer cells. While the relationship between chemotherapy and epigenetics has been widely discussed, emerging evidence indicates that specific epigenetic effectors are also crucial for the development of resistance to tyrosine kinase inhibitors (TKIs). One particular gene that encodes the histone lysine demethylase KDM5A is overexpressed in several cancers. In breast cancer tissues, cells with KDM5A gene amplification were found to be more resistant to erlotinib, an inhibitor of the tyrosine kinase epidermal growth factor receptor (EGFR), when compared to cells without the same amplification. KDM5A was also shown to mediate resistance to a second EGFR inhibitor called gefitinib, in EGFR-mutant lung cancer cell lines. This evidence indicates that KDM5A could activate alternative survival pathways involved in overcoming EGFR inhibition. In line with these results, another histone demethylase (i.e., KDM1A) promotes liver cancer cells’ resistance to the TKI sorafenib. Current evidence provides a suitable rationale to consider the use of specific KDMs inhibitors to sensitize cells to tyrosine kinase targeted therapies and thus, presents an opportunity to prevent the further development of drug resistance. This review discusses the involvement of histone lysine demethylases in the development of resistance to TKI and highlights the importance to develop new cancer treatment regimens to counteract this phenomenon.

Disruption of the Interaction of RAS with PI 3-Kinase Induces Regression of EGFR-Mutant-Driven Lung Cancer

SummaryRAS family GTPases contribute directly to the regulation of type I phosphoinositide 3-kinases (PI3Ks) via RAS-binding domains in the PI3K catalytic p110 subunits. Disruption of this domain of p110α impairs RAS-mutant-oncogene-driven tumor formation and maintenance. Here, we test the effect of blocking the interaction of RAS with p110α on epidermal growth factor receptor (EGFR)-mutant-driven lung tumorigenesis. Disrupting the RAS-PI3K interaction inhibits activation of both AKT and RAC1 in EGFR-mutant lung cancer cells, leading to reduced growth and survival, and inhibits EGFR-mutant-induced tumor onset and promotes major regression of established tumors in an autochthonous mouse model of EGFR-mutant-induced lung adenocarcinoma. The RAS-PI3K interaction is thus an important signaling node and potential therapeutic target in EGFR-mutant lung cancer, even though RAS oncogenes are not themselves mutated in this setting, suggesting different strategies for tackling tyrosine kinase inhibitor resistance in lung cancer.

Increased Synthesis of MCL-1 Protein Underlies Initial Survival of EGFR-Mutant Lung Cancer to EGFR Inhibitors and Provides a Novel Drug Target.

Purpose: EGFR inhibitors (EGFRi) are effective against EGFR-mutant lung cancers. The efficacy of these drugs, however, is mitigated by the outgrowth of resistant cells, most often driven by a secondary acquired mutation in EGFR, T790M We recently demonstrated that T790M can arise de novo during treatment; it follows that one potential therapeutic strategy to thwart resistance would be identifying and eliminating these cells [referred to as drug-tolerant cells (DTC)] prior to acquiring secondary mutations like T790M Experimental Design: We have developed DTCs to EGFRi in EGFR-mutant lung cancer cell lines. Subsequent analyses of DTCs included RNA-seq, high-content microscopy, and protein translational assays. Based on these results, we tested the ability of MCL-1 BH3 mimetics to combine with EGFR inhibitors to eliminate DTCs and shrink EGFR-mutant lung cancer tumors in vivo Results: We demonstrate surviving EGFR-mutant lung cancer cells upregulate the antiapoptotic protein MCL-1 in response to short-term EGFRi treatment. Mechanistically, DTCs undergo a protein biosynthesis enrichment resulting in increased mTORC1-mediated mRNA translation of MCL-1, revealing a novel mechanism in which lung cancer cells adapt to short-term pressures of apoptosis-inducing kinase inhibitors. Moreover, MCL-1 is a key molecule governing the emergence of early EGFR-mutant DTCs to EGFRi, and we demonstrate it can be effectively cotargeted with clinically emerging MCL-1 inhibitors both in vitro and in vivo Conclusions: Altogether, these data reveal that this novel therapeutic combination may delay the acquisition of secondary mutations, therefore prolonging therapy efficacy. Clin Cancer Res; 24(22); 5658-72. ©2018 AACR.

The different path of T790M-positive EGFR-mutant lung cancer

Approximately half of the cases of acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are mediated by the secondary T790M mutation (1,2). Distinct clinical course of patients with T790M-positive lung cancer has been suggested, mostly in the context of more indolent progression and favorable prognosis (3,4). It is in line with the pre-clinical study showing that EGFR-mutant cell lines with acquired T790M mutation exhibit slower growth although the way how the additional T790M mutation can affect the growth rate of EGFR-mutant lung cancer cells remains unclear (5).

P1.13-10 Aurora Kinase A Drives the Evolution of Resistance to Third Generation EGFR Inhibitors in Lung Cancer

Paradigm defining for precision medicine, EGFR inhibitors are a major breakthrough in the treatment of EGFR-mutant non-small cell lung cancer (NSCLC). Although these EGFR-TKI therapies often elicit profound initial therapeutic responses, their effects are transient due to residual disease. This residual disease and subsequent disease progression occurs through tumor evolution and molecular drivers behind the formation, maintenance and evolution of residual disease and acquired resistance have remained elusive. Although in many cases pre-existing clones with bona-fide genetic resistance have been identified, majority of patients have undetectable resistance causing genetic alterations suggesting that non-genetic alterations may drive altered cell state and signaling associated with EGFR inhibitor resistance. Furthermore, in the case of osimertinib now used in first line setting, mechanisms of acquired resistance have been ill defined and no effective second line therapies exist. Using EGFR mutant lung cancer cells we developed eight distinct in vitro models of acquired resistance to 3rd generation EGFR-TKI inhibitors, osimertinib and rociletinib. We examined the efficacy of drug combinations in vivo using both cell line xenografts and PDX models of EGFR TKI resistance. We measured the level of staining of the candidate biomarker, TPX2, in patients progressing on first and third-generation EGFR TKIs. Chemical screens in acquired resistant cell lines revealed that Aurora kinase inhibitors are highly synergistic when combined with third-generation EGFR inhibitors. Resistant cells harbored heightened activation of AURKA caused by upregulation of its co-activator protein TPX2. In in-vitro and in-vivo models of acquired resistance the combination induced potent cell death by reactivating BIM-mediated apoptosis. We found that tumors from the majority of patients progressing on first and third-generation EGFR TKIs harbored high levels of TPX2, indicating that AURKA is likely activated and driving resistance in a significant fraction of EGFR-mutant lung cancers. Tracking the kinetics of AURKA activation, we found that AURKA activity is required for the formation and maintenance of residual drug tolerant cells, precursors of acquired resistance. Either single agent EGFR-TKI, the AURKA inhibitor MLN8237 or the combination enhanced the magnitude of response and forestalled the emergence of resistance in vitro as compared to monotherapies. The combination robustly induced tumor regressions in an EGFR L858R PDX tumor model generated from a residual disease surgical specimen. This synthetic lethal interaction between EGFR TKIs and Aurora kinase inhibitors has important clinical implications for the development of better treatment strategies using EGFR-TKIs and suggest a new paradigm for preventing the emergence of resistance.

Abstract PR13: MET copy number gain is associated with gefitinib resistance in leptomeningeal carcinomatosis of EGFR-mutant lung cancer

Abstract Leptomeningeal carcinomatosis occurs frequently in EGFR-mutant lung cancer, and develops acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs). This study aimed to clarify the mechanism of EGFR-TKI resistance in leptomeningeal carcinomatosis and seek a novel therapeutic strategy. We examined EGFR mutations, including the T790M gatekeeper mutation, in 32 re-biopsy specimens from 12 leptomeningeal carcinomatosis and 20 extracranial lesions of EGFR-mutant lung cancer patients who became refractory to EGFR-TKI treatment. All 32 specimens had the same baseline EGFR mutations, but the T790M mutation was less frequent in leptomeningeal carcinomatosis specimens than in extracranial specimens (8% vs. 55%, P < 0.01). To study molecular mechanisms of acquired EGFR-TKI resistance in leptomeningeal carcinomatosis, we utilized our previously developed mouse model of leptomeningeal carcinomatosis with the EGFR-mutant lung cancer cell line PC-9/ffluc cells, in which acquired resistance to gefitinib was induced by continuous oral treatment. Compared with subcutaneously inoculated gefitinib-resistant tumors, the T790M mutation was less frequent in leptomeningeal carcinomatosis that acquired resistance to gefitinib. PC-9/LMC-GR cells were established from the gefitinib-resistant leptomeningeal carcinomatosis model, and they were found to be intermediately resistant to gefitinib and osimertinib (third-generation EGFR-TKI). Although EGFR-T790M was negative, gefitinib resistance of PC-9/LMC-GR cells was related to MET copy number gain with MET activation. Moreover, combined use of EGFR-TKI and crizotinib, a MET inhibitor, dramatically regressed leptomeningeal carcinomatosis with acquired resistance to gefitinib or osimertinib. These findings suggest that combination therapy with MET inhibitors may be promising for controlling leptomeningeal carcinomatosis that acquires resistance to EGFR-TKIs. This abstract is also being presented as Poster B29. Citation Format: Shigeki Nanjo, Sachiko Arai, Wei Wang, Akito Hata, Nobuyuki Katakami, Seiji Yano. MET copy number gain is associated with gefitinib resistance in leptomeningeal carcinomatosis of EGFR-mutant lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR13.

Abstract 4156: Inhibition of NF-kB improves sensitivity to radiation and EGFR inhibitor and decreases radiation-induced lung toxicity in lung cancer

Abstract Combination with EGFR-TKIs and radiation seems to be a good strategy against lung cancer, since radiation could enhance EGFR expression. However, the results of clinical trials were controversial. MET activation was involved in resistance to both radiation and EGFR inhibitor, while NF-kB was reported to closely relate to HGF/MET signaling axis. Furthermore, NF-κB has been proved to play a role in bleomycin-induced pulmonary fibrosis, which could be ameliorated by IKKB inhibitor (IMD-0354). Therefore, we evaluated the influence of NF-kB/MET inhibition to the sensitivity of radiation and EGFR-TKIs in lung cancer cells in vitro and in vivo. we also examined the role of NF-κB in radiation-induced lung toxicity. Our data showed that radiation enhanced activation and expression of MET and EGFR, whereas only activation of MET could be reversed by IMD-0354 and p65 depletion, suggesting that radiation-induced MET activation was mediated by NF-kB signaling. Furthermore, NF-kB inhibition increased radiation-induced DNA damage and apoptosis in lung cancer cells. On the other hand, radiation decreased sensitivity to EGFR inhibitor in EGFR mutant lung cancer cells, while inhibition of NF-kB or MET overcame the resistance. Importantly, IMD-0354 increased sensitivity to radiation, EGFR-TKIs, or their combination in mouse xenograft tumors by arresting cell proliferation and inducing cell apoptosis. Interestingly, IMD-0354 significantly reduced the lung toxicity in a mouse model of radiation-induced pneumonia and lung fibrosis. Our preclinical findings indicated that inhibition of NF-kB/MET could improve sensitivity to radiation and EGFR inhibitor and decrease radiation-induced lung toxicity in lung cancer. Citation Format: Wei Wang, Rong Wang, Shunli Peng, Qi Li, Xiaojuan Zhang, Yueyun Ma. Inhibition of NF-kB improves sensitivity to radiation and EGFR inhibitor and decreases radiation-induced lung toxicity in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4156.